This invention is in the field of bacterial plasmids. More specifically, a bacterial plasmid has been isolated which contains genes necessary for plasmid maintenance and for degrading aromatic compounds to small aliphatic molecules.
It is well known that bacterial genes are sometimes located on plasmids (Actis et al., Front. Biosci. 4:D43-62 (1999); del Solar et al., Microbiol. Mol. Biol. Rev. 62:434-464 (1998)). Plasmids are not necessary for routine xe2x80x9chousekeepingxe2x80x9d functions in bacteria (e.g., DNA synthesis and protein synthesis). However, the genes on plasmids are often important in specialized environments. Antibiotic resistance genes and heavy metal resistance genes are examples of genes commonly found on plasmids. Although plasmids are similar in function to chromosomes as carriers of genes, plasmids can be distinguished from chromosomes. Plasmids are smaller than chromosomes and encode functions that are dedicated to plasmid replication. Each type of plasmid has a distinct ori sequence and rep gene(s) for initiation of replication. These sequences constitute the minimum requirements for a functional, replicating plasmid. Fu et al. (Mol Gen Genet 250:699-704 (1996); Plasmid 38:141-147 (1997)) has reported RepA protein that has replication initiation and transcription repression function.
The genetic information for degradation of aromatic chemicals is also frequently located on plasmids in bacteria (Assinder and Williams, Adv. Microb. Physiol. 31:2-69 (1990)). The well-characterized TOL plasmid pWWO is typical of many bacterial plasmids that have genes for degradation of aromatic compounds (Assinder and Williams, Adv. Microb. Physiol. 31:2-69 (1990)). Plasmid pWWO is 117 kb in size, is transmissible, and belongs to the broad host range IncP-9 incompatibility group of plasmids. The pWWO xyl genes encode enzymes for metabolism of toluene, m-xylene, and p-xylene.
Understanding the make-up of bacterial plasmids derived form species known to participate in degradative reactions is key to the design of more effective degrading species. Because of their ease of transmission, bacterial plasmids are useful tools for moving degradative genes into hosts. Additionally, because they are so promiscuous, many plasmids carry genes that have been adapted for expression in hosts other than the species from which the plasmid has been isolated.
In spite of the utility of bacterial plasmids, few have been fully characterized. There remains a need therefor for bacterial plasmids comprising genes encoding degradative enzymes whose replication and stability functions are understood and may be manipulated for the broad spectrum expression of degradative genes. Applicants have met this need by the discovery and isolation of a plasmid, CT14 from a Pseudomonas sp. carrying genes necessary for replication and stability in a host as well as genes encoding enzymes for the degradation of various aromatic substrates including catechols and other intermediates in the toluene degradative pathway.
The present invention provides an isolated nucleic acid fragment encoding a bacterial aromatic ring opening enzyme selected from the group consisting of: (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:10; (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:10; (c) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1xc3x97SSC, 0.1% SDS, 65xc2x0 C. and washed with 2xc3x97SSC, 0.1% SDS followed by 0.1xc3x97SSC, 0.1% SDS; and (d) an isolated nucleic acid fragment that is complementary to (a), (b) or (c). Preferred nucleic acid fragments are those that have about 90% identity to the nucleic acid fragment selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:9. The present invention additionally provides polypeptides encoded by the isolated nucleic acid fragment of the bacterial aromatic ring opening genes.
The invention also provides a method of obtaining a nucleic acid fragment encoding all or a substantial portion of a bacterial aromatic ring opening enzyme comprising: (a) probing a genomic library with the nucleic acid fragment encoding a bacterial aromatic ring opening enzyme; (b) identifying a DNA clone that hybridizes with the nucleic acid fragment encoding a bacterial aromatic ring opening enzyme under the following conditions; 0.1xc3x97SSC, 0.1% SDS, 65xc2x0 C. and washed with 2xc3x97SSC, 0.1% SDS followed by 0.1xc3x97SSC, 0.1% SDS; and (c) optionally sequencing the genomic fragment that comprises the clone identified in step (b), wherein the sequenced genomic fragment encodes all or substantially all of an amino acid sequence encoding a bacterial aromatic ring opening enzyme.
Alternatively the present invention provides a method of obtaining a nucleic acid fragment encoding all or a substantial portion a bacterial aromatic ring opening enzyme comprising: (a) synthesizing at least one oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; and (b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a); wherein the amplified insert encodes a portion of an amino acid sequence encoding a bacterial aromatic ring opening enzyme.
The present invention additionally provides an isolated nucleic acid fragment encoding a bacterial glutathione-S transferase selected from the group consisting of: (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO:8; (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth SEQ ID NO:8; (c) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1xc3x97SSC, 0.1% SDS, 65xc2x0 C. and washed with 2xc3x97SSC, 0.1% SDS followed by 0.1xc3x97SSC, 0.1% SDS; (d) an isolated nucleic acid fragment having at least 69% identity with the amino acid as set forth in SEQ ID NO:8; and (e) an isolated nucleic acid fragment that is complementary to (a), (b), (c) or (d). The invention additionally provides methods for the obtaining nucleic acid fragment encoding all or a substantial portion of a bacterial glutathione-S transferase protein by method of hybridization or primer directed amplification. Additionally, the present invention provides polypeptides encoded by the isolated nucleic acid fragment of the GST genes.
In similar fashion the invention provides an isolated nucleic acid fragment encoding a bacterial plasmid maintenance protein selected from the group consisting of: (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:22; (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:22; (c) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1xc3x97SSC, 0.1% SDS, 65xc2x0 C. and washed with 2xc3x97SSC, 0.1% SDS followed by 0.1xc3x97SSC, 0.1% SDS; and (d) an isolated nucleic acid fragment that is complementary to (a), (b) or (c). Preferred nucleic acids encoding a bacterial plasmid conjugation protein are those having 90% identity to the nucleic acid fragment selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21. Methods for obtaining a nucleic acid fragment encoding all or a substantial portion of a bacterial plasmid maintenance protein are also provided employing nucleic acid hybridization or primer directed amplification. The present invention additionally provides polypeptides encoded by the isolated nucleic acid fragment of the bacterial plasmid maintenance proteins.
The present invention additionally provides chimeric genes comprising suitable regulatory sequences operably linked to the instant sequences. The chimeric genes may be used of transform suitable host cells including bacteria, yeast, and filamentous fungi.
The invention further provides a method for the degradation of a catechol substrate comprising contacting a transformed host cell under suitable growth conditions with an effective amount of catechol substrate whereby the catechol substrate is degraded, said transformed host cell comprising a nucleic acid fragment encoding a bacterial catechol dioxygenase as set forth in SEQ ID NO:4 under the control of suitable regulatory sequences.
Additionally the invention provides a method for the degradation of a semialdehyde substrate comprising contacting a transformed host cell under suitable growth conditions with an effective amount of catechol substrate whereby the semialdehyde substrate is degraded, said transformed host cell comprising a nucleic acid fragment encoding a dehydrogenase as set forth in SEQ ID NO:6 under the control of suitable regulatory sequences.
In similar fashion the invention provides a method for the degradation of a substrate selected from the group consisting of 2-hydroxypent-2,4-dienoate, methyl-2-hydroxymuconic-semialdehyde and chloro-2-hydroxymuconic-semialdehyde comprising contacting a transformed host cell under suitable growth conditions with an effective amount of said substrate whereby the substrate is degraded, said transformed host cell comprising a nucleic acid fragment encoding a dehydrogenase as set forth in SEQ ID NO:10 under the control of suitable regulatory sequences.
The invention further provides the bacterial strain CT14 comprising the plasmid pCT14. The bacterial strain CT14 is useful in a method for the degradation of aromatic compounds comprising contacting the strain with an aromatic compound under suitable growth conditions wherein the aromatic compounds is degraded. Similarly CT14 is useful in a method of reducing the level of mercury in a mercury contaminated environment comprising a cell containing the plasmid pCT14 under suitable growth conditions whereby the level of mercury in the contaminate environment is reduced.